scholarly journals MICROFILAMENTS AND CELL LOCOMOTION

1971 ◽  
Vol 49 (3) ◽  
pp. 595-613 ◽  
Author(s):  
Brian S. Spooner ◽  
Kenneth M. Yamada ◽  
Norman K. Wessells
Keyword(s):  
Protein Synthesis ◽  
Plasma Membrane ◽  
Cytochalasin B ◽  
Cell Movement ◽  
Leading Edge ◽  
Complete Recovery ◽  
Cell Locomotion ◽  
Cell Processes

The role of microfilaments in generating cell locomotion has been investigated in glial cells migrating in vitro. Such cells are found to contain two types of microfilament systems: First, a sheath of 50–70-A in diameter filaments is present in the cytoplasm at the base of the cells, just inside the plasma membrane, and in cell processes. Second, a network of 50-A in diameter filaments is found just beneath the plasma membrane at the leading edge (undulating membrane locomotory organelle) and along the sides of the cell. The drug, cytochalasin B, causes a rapid cessation of migration and a disruption of the microfilament network. Other organelles, including the microfilament sheath and microtubules, are unaltered by the drug, and protein synthesis is not inhibited. Removal of cytochalasin results in complete recovery of migratory capabilities, even in the absence of virtually all protein synthesis. Colchicine, at levels sufficient to disrupt all microtubules, has no effect on undulating membrane activity, on net cell movement, or on microfilament integrity. The microfilament network is, therefore, indispensable for locomotion.

scholarly journals The role of extracellular materials in cell movement. I. Inhibition of mucopolysaccharide synthesis does not stop ruffling membrane activity or cell movement.

1975 ◽  
Vol 65 (2) ◽  
pp. 286-297 ◽  
Author(s):  
B S Spooner ◽  
G W Conrad
Keyword(s):  
Cytochalasin B ◽  
Cell Movement ◽  
Embryonic Heart ◽  
Membrane Activity ◽  
Cell Locomotion ◽  
Extracellular Materials

The involvement of mucopolysaccharide synthesis in cell locomotion was investigated by determining the effects of inhibition of synthesis on ruffling membrane activity and cell movement by embryonic heart fibroblasts. Mucopolysaccharide synthesis was inhibited directly by treatment with a glutamine analog, 6-diazo-5-OXO-L-norleucine (DON), and indirectly with cycloheximide. DON treatment reduced synthesis to 20% of control values, and cycloheximide reduced synthesis to less than 10% of control values, as measured by incorporation of [35S]sulfate into mucopolysaccharides. Nevertheless, ruffling membrane activity and cell locomotion continued under both conditions. Cytochalasin B did not inhibit mucopolysaccharide synthesis, although it did stop ruffling and locomotion. These results suggest that if mucopolysaccharides are required for cell movement, they must have long half-lives or represent only a minute fraction of the normal synthetic load.

Rho GTPase CDC42 Signals Separately Regulate Directed Migration Versus Random Movement in Neutrophil.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 636-636
Author(s):  
Marie-Dominique Filippi ◽  
Haiming Xu ◽  
Kathleen Szczur ◽  
Yi Zheng ◽  
David A. Williams
Keyword(s):  
Rho Gtpases ◽  
Cell Movement ◽  
Leading Edge ◽  
Actin Assembly ◽  
Directed Migration ◽  
Attachment Structures ◽  
Pmn Functions

Abstract Neutrophils (PMN) are a critical cell in inflammation. In response to external stimuli, they activate various signaling pathways to move rapidly to a site of microbial invasion and perform phagocytosis, cytokine and reactive oxygen species release. Rho GTPases, Rac1, Rac2, CDC42 and Rho, are central regulators of cell movement via actin rearrangement. We have shown the specific role of Rac1 and Rac2 in PMN functions (Gu, Science 2003; Filippi, Nat Immunol, 2004) which raises the question of the role of other Rho GTPases in PMN functions. CDC42 primarily regulates filopodia formation and controls cell polarity and migration in non-hematopoietic cells and some hematopoietic cell lines. Most of previous studies have used dominant active or negative mutants which lack specificity and cannot be used to define in vivo cell biology. Here, we used mice genetically deficient in the CDC42 negative regulator CDC42 GTPase Activating Protein (GAP) to study the role of CDC42 in PMN functions in vitro and in vivo. PMN deficient in CDC42GAP (CDC42GAP−/−) displayed a 2-fold increase in CDC42 activity. In vivo recruitment of PMN in peritoneal cavities was significantly higher in CDC42GAP−/− animals than in WT mice (4.5 ± 0.3x106 vs 3.4 ± 0.2x106, p<0.05) indicating that CDC42 plays a physiological role in neutrophil migration. We examined F-actin assembly upon integrin ligation. Podosome-like structures identified by a vinculin ring surrounding F-actin that are present at the leading edge in WT PMN were significantly reduced in frequency in the mutant cells (15% vs 3%). In addition, CDC42GAP−/− PMN showed increased lateral filopodia-like formation and abnormally elongated uropod with tail filopodia. Thus, CDC42GAP−/− PMN appeared less polarized than WT PMN (50% vs 16%). This abnormal F-actin assembly was associated with abnormal cell motility. In vitro, CDC42GAP−/− PMN showed increase random movement (chemokinesis) compared with WT PMN. By contrast but similar to the loss of CDC42 activity, CDC42GAP−/− PMN displayed defective directed migration towards fMLP suggesting that CDC42 activity plays a critical role in both chemokinesis and directed migration. These functions may be regulated by podosome-like and filopodia formation respectively. To further understand this correlation at a mechanistic level, we examined MAPK signaling. CDC42GAP−/− PMN showed sustained ERK phosphorylation at 15min compared to WT PMN. By contrast, p38MAPK was significantly decreased in CDC42GAP−/− PMN compared to WT at both 5 and 15min. Pharmacological inhibition of ERK activity in CDC42GAP−/− PMN using U0126 rescued the abnormal increased chemokinesis to level similar to WT and was associated with partial rescue of podosome-like formation at the leading edge of the cells. Inhibition of p38MAPK activity in WT PMN using SB203580 reduced directed migration and was associated with increased tail filopodia that mimicked CDC42GAP−/− PMN. Taken together, these results suggest that CDC42GAP plays an important role in PMN chemokinesis and directed migration likely via distinct signaling pathways. CDC42GAP may control chemokinesis via ERK-mediated podosome-like turnover at the leading edge. CDC42GAP may regulate directed migration by inhibiting filopodia at the uropod via p38MAPK and subsequently by restraining filopodia to the leading edge. This reinforces the importance of turnover of attachment structures during cell movement and suggests a new role for CDC42 in attachment structures in neutrophils and for p38MAPK in CDC42-mediated directed migration.

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

2021 ◽  
Author(s):  
Cecilia Valencia ◽  
Felipe Alonso Pérez ◽  
Carola Matus ◽  
Ricardo Felmer ◽  
María Elena Arias
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Protein Synthesis Inhibitors ◽  
Vitro Fertilization ◽  
New Findings ◽  
Bovine Oocytes ◽  
Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

scholarly journals Protein Synthesis in Human Leukocytes and Lymphocytes: 1. Effect of Steroids and Sterols

Blood ◽  
1969 ◽  
Vol 34 (3) ◽  
pp. 348-356 ◽  
Author(s):  
SEYMOUR WERTHAMER ◽  
CARL HICKS ◽  
LEONARD AMARAL
Keyword(s):  
Protein Synthesis ◽  
Steroid Hormones ◽  
Plasma Protein ◽  
Human Lymphocytes ◽  
Amino Acid Incorporation ◽  
Binding Factor ◽  
In Vitro Effects ◽  
Physiologic Role

Abstract The in vitro effects of sterols, cholesterol and 3-methyl cholanthrene and steroids, cortisol, prednisolone and testosterone on protein synthesis in separate popultions of human lymphocytes and leukocytes has been investigated. It has been shown that all agents used result in the inhibition of protein synthesis under these conditions. It has also been shown that the inhibitory mechanism of the steroid hormones requires the presence of plasma, presumably as a protein binding factor in order to achieve its effect. The sterol, cholesterol and 3-methyl cholanthrene, in the absence of plasma, still inhibit amino acid incorporation. However, in the case of cholesterol, the magnitude of inhibition is lower than that observed in the presence of plasma, perhaps indicating a partial plasma dependence. The results presented therefore support the hypothesis that the inhibition of lymphocyte protein synthesis by steroid hormones occurs only when the steroid is bound to a plasma protein. The physiologic role of the plasma protein-cortisol complex and its relation to the condition of lymphopenia in man is discussed.

scholarly journals Purinergic Modulation of Interleukin-1β Release from Microglial Cells Stimulated with Bacterial Endotoxin

1997 ◽  
Vol 185 (3) ◽  
pp. 579-582 ◽  
Author(s):  
Davide Ferrari ◽  
Paola Chiozzi ◽  
Simonetta Falzoni ◽  
Stefania Hanau ◽  
Francesco Di  Virgilio
Keyword(s):  
Plasma Membrane ◽  
P2x7 Receptor ◽  
Purinergic Receptor ◽  
Present Report ◽  
Physiological Role ◽  
Bacterial Endotoxin ◽  
Microglial Cells

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today. 16:524–528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R.A. North and G. Buell. 1996. Science (Wash. DC). 272:735–737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1β. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1β release triggered by LPS. Our data suggest that LPS-dependent IL-1β release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1β secretion.

Heat-shock-induced grey crescent formation in axolotl eggs and oocytes: the role of gravity

Development ◽  
1987 ◽  
Vol 100 (4) ◽  
pp. 599-609
Author(s):  
J.-C. Beetschen ◽  
J. Gautier
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Synergistic Action ◽  
Nuclear Factor ◽  
Crescent Formation ◽  
Animal Pole ◽  
Grey Crescent ◽  
Cytoskeletal Structure

Axolotl eggs were heat shocked (36.8°C, 10min) inside their jelly layers. Heat shock (HS) was shown to induce the precocious appearance of a grey crescent (GC) in a number of eggs immediately after fertilization (Benford & Namenwirth, 1974). It was also demonstrated that this phenomenon occurs in fertilized or artificially activated eggs only when they are shocked within 11/2h after spawning. The GC forms still later in heated unfertilized, nonactivated eggs. The role of the jelly layers is considered to be mechanical: a proportion of eggs is maintained in a tilted position until the egg is able to orient animal pole upwards under the influence of gravity as a late consequence of activation. The jelly layers are not essential if the eggs are artificially tilted or rotated during HS. GC formation can also be induced in in vitro maturing oocytes, provided they are tilted during HS. Gravity thus plays an essential role in the cytoplasmic rearrangements leading to HS-induced GC formation. Our results indicate a synergistic action between heat and gravity in this process. The cytological appearance of the GC formed in those experiments is that of a ‘Born's crescent’ with a conspicuous ‘vitelline wall’ (Pasteels, 1964). When oocytes are enucleated before maturation, HS has no effect on GC formation. A nuclear factor is therefore essential, as has been demonstrated in early GC formation induced by inhibitors of protein synthesis. Finally, incorporation of amino acids into oocyte proteins appears to be rapidly inhibited by HS (from 5 min). However, we cannot conclude that GC formation is in fact triggered by inhibition of protein synthesis. It is also likely that HS disrupts cytoskeletal structure, hence facilitating cytoplasmic rearrangements. Nevertheless, these results are in agreement with the scheme we recently proposed for GC formation in the rotated axolotl oocyte (Gautier & Beetschen, 1985).

Multinucleated rat alveolar macrophages express functional receptors for calcitonin

1991 ◽  
Vol 261 (6) ◽  
pp. F1026-F1032 ◽  
Author(s):  
A. Vignery ◽  
M. J. Raymond ◽  
H. Y. Qian ◽  
F. Wang ◽  
S. A. Rosenzweig
Keyword(s):  
Plasma Membrane ◽  
Alveolar Macrophages ◽  
Giant Cells ◽  
Mononuclear Phagocytes ◽  
Inflammatory Reactions ◽  
Calcitonin Gene ◽  
Novel Model

The fusion of mononuclear phagocytes occurs spontaneously in vivo and leads to the differentiation of either multinucleated giant cells or osteoclasts in chronic inflammatory sites or in bone, respectively. Although osteoclasts are responsible for resorbing bone, the functional role of giant cells in chronic inflammatory reactions and tumors remains poorly understood. We recently reported that the plasma membrane of multinucleated macrophages is, like that of osteoclasts, enriched in Na-K-adenosinetriphosphatases (ATPases). We also observed that the localization of their Na-K-ATPases is restricted to the nonadherent domain of the plasma membrane of cells both in vivo and in vitro, thus imposing a functional polarity on their organization. By following this observation, we wished to investigate whether these cells also expressed, like osteoclasts, functional receptors for calcitonin (CT). To this end, alveolar macrophages were fused in vitro, and both their structural and functional association with CT was analyzed and compared with those of mononucleated peritoneal and alveolar macrophages. Evidence is presented that multinucleated alveolar macrophages express a high copy number of functional receptors for CT. Our results also indicate that alveolar macrophages, much like peritoneal, express functional receptors for calcitonin gene-related peptide. It is suggested that multinucleated rat alveolar macrophages offer a novel model system to study CT receptors and that calcitonin may control local immune reactions where giant cells differentiate.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation

1996 ◽  
Vol 43 (2) ◽  
pp. 228-235 ◽  
Author(s):  
Li Meng ◽  
Jean Rutledge ◽  
Ying Zhu ◽  
Gerald M. Kidder ◽  
Firouz Khamsi ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
In Vitro Maturation ◽  
Germinal Vesicle
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